JOURNAL OF VIROLOGY, Oct. 1977, p.211-221 Copyright© 1977 American Society forMicrobiology
Vol. 24, No. 1 Printedin U.S.A.
Specific Binding Sites for a Parvovirus, Minute Virus of Mice,
on
Cultured Mouse Cells
P. LINSER,* HELEN BRUNING, AND R. W. ARMENTROUT
Department ofBiological Chemistry, University of Cincinnati Medical Center, Cincinnati, Ohio 45267 Received forpublication 30March1977
The early
interactions between parvovirusesand host cells have
not beenextensively described previously.
Inthis
study
we have characterized someaspects
of
viral
binding
tothe cell
surface
and demonstrated the existence ofspecific cellular
receptorsites for minute virus
of mice (MVM) on two murinecell lines that
are permissivefor viral growth.
The interaction had a pHoptimum
of
7.0 to7.2,and both the
rateand
extentof the reactions
wereslightlyaffected by
temperature. Mouse A-9 cells (L-cell derivative) had -5 x 105specific
MVM
binding
sites percell,
and Friend erythroleukemia cells had
1.5 x 105MVM
sites per cell. In contrast, thenonpermissive
mouselymphoid cell
lineL1210
lacked specific viral
receptors.Also,
cloned lines of A-9
cells resistant
toviral infection have been isolated. One
of these lineslacked the
"specific"
virusattachment sites but exhibited low levels of nonsaturable
virusbinding.
Basedon
these examples, infectivity
iscorrelated with the
presenceof
specific viral
receptors on
the cell surface.
We
have looked
atthe initial interactionbe-tween
minute
virusof
mice(MVM)
and several
murine tissue
culture
celllines
inorder
tochar-acterize
and quantify
aspecific
virus-binding
site. MVM
is a parvovirus, aclass of small
viruses that contain a linear single-stranded
DNA
genome.Very little information concern-ing early parvovirus host cell interactions hasbeen
previously reported
(19).However,
the
binding of
picornaviruses tocells has
beenex-tensively studied and
is in some respectsanalo-gous to
the
parvovirus system. Parvovirusesand
picornaviruses
are roughly the same size(-17 to 30 nm in
diameter), and they both
consist
of
anicosahedral
proteincapsid
sur-rounding
asingle-stranded nucleic acid
genome(RNA
inthe
caseof
picornaviruses).The initial
interaction
of
picornaviruses such as polioviruswith
susceptible host cells has been
character-ized. The
number of specific binding sites percell is known (-104/cell)
(10, 14, 15),and
thepresence
of
specific cellular binding
sites in partexplains
the
cytological specificity of
polio-virus infections. There
isevidence thatparvovi-ruses may be tissuespecific as well (3, 12), and
this
report is the first evidence for arelation-ship
betweeninfectivity
and the presence ofspecific virus-binding
sites on the host cell.MATERIALS AND METHODS
Virus stocks. All cell lines were free of
myco-plasmas determined periodically by
autoradiogra-phy. Plaque-purified MVM wasthegenerousgiftof 211
Peter Tattersall. MVM labeled in its DNA with
[methyl-3H]thymidine wasgrowninRT-7cells (pre-viously described[18]). Randomlygrowinginfected cells were labeled for 48 h during the interval of maximumviral encapsulation. Half-confluent mon-olayers of RT-7 cells in Corning T-flasks were in-fected with MVMpurifiedfrom the110Sregion ofa
sucrosegradient. The virus was adsorbedtocells in phosphate-buffered saline(PBS) at37°C for2h.The monolayers were then fed withfresh F-11(minimal
essential medium, Grand Island Biological Co.
[GIBCOQ)
with5%heat-inactivated fetal calfserumand 100U ofpenicillin and100 ,ug ofstreptomycin per ml.Twenty-fourhours after infection the mono-layers weresubcultured and refed with fresh
me-dium containing25,uCi of[methyl-3Hlthymidineper ml. Forty-eight hours later the monolayers were harvested into 0.01 M Tris buffer (pH 9.0),disrupted
by sonic treatment, andextractedtwicewith5 vol-umesof Freon. The aqueousphasewasbroughtto a
final concentration of 10 mM
MgCl2.
DNase(Worth-ingtonBiochemicals Corp.) wasadded to0.1mg/ml, and themixture wasincubatedina37°Cwaterbath for60min.Thecrude preparationwasthendialyzed
at4°Covernightagainst3x 1-literchangesof0.01M Tris-0.005 M EDTA (pH 9.0) buffer. The preparation was thenlayered onto 37-ml continuous 15 to 30%
sucrosegradientsandcentrifugedinanSW-27 rotor at25,000 rpm and4°Cfor 6.5 h. The110Speakoffull
virus was located by a combination of the optical density at 260nm,thehemagglutinin activity, and the DNase-resistant, acid-insoluble radioactivity assayed across the gradientaspreviously described (18). The concentration of virus particlesinthe virus preparationswasmeasuredby the opticaldensityat 280 nm and calculated from an El% of71.2 as de-scribed by Tattersall et al. (21). Essentially the
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sameresults were obtained when the -concentration ofparticles wasestimated from the protein concen-tration as determined by fluorescence (5) and using a value of 4 x 106 daltons of protein per mol of particles. Infectivity of preparations of this type is typically 1 infectious unit per 4 x 102 to 6 x 102 particles, as measured by 50% tissue culture infec-tivedose or plaque assay.
A-9 cells, a suspension culture derivative of mouse L-cells (13), were grown in F-11 (minimal essential medium, GIBCO)supplemented with 10% heat-inactivated fetal calf serum and 100 U of peni-cillin and 100 ,ug of streptomycin per ml in suspen-sion atdensities from 5 x 104 to 5 x 105/ml. These cells are permissive for viral growth (20) and repre-sentthe model system used to define several param-etersof virus-cell binding in this study.
Murine Friend-745 erythroleukemia cells (Friend virus-transformed spleen cells) (6, 8) were provided by K. Lowenhaupt. These cells are permissive for viral growth (16a) and can be induced to un-dergo erythropoietic "differentiation" in culture
bydimethyl sulfoxide (Me2SO)treatment (6). These cells are suspension adapted and were grown in Ham F-12 (GIBCO) supplemented with 10% fetal calf serum at suspensiondensities of2 x 104to4 x
105/ml. These cellswere inducedby the addition of 1.8%Me2SO to 4 x 104 cells per mlin fresh F-12. The course ofinduction was monitored by scoring the percentage of cells that were positive for hemoglobin by the benzidine staining technique (6).
The mouse lymphoid cell line L1210 was the gift of J. McCormick. These cells do not support MVM growth (16a) andconstitute the negative control for virus binding used in this study. L1210 cells are also grown in suspension in RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated fetal calf
serumatdensities of 2 x 104 to 5 x 105/ml. Binding assay. Binding ofradiolabeled virus to cells was performed in suspension with periodic gentle mixing to keep cells suspended. Reactions werecarried out in sterile Corning disposable poly-styrene conical centrifuge tubes, as described for each experimentinResults. Unless otherwise
speci-fied,for eachdata point a known quantity of virus in
asmall volume (i.e.,5to 50 ul) was addedto 2 x 105
cells in1ml of buffer. The virus wasstored at-200C
in the sucrose solution of the isolation gradient, conditions which prevented viral aggregation. After the appropriate incubation time, the sample was filtered through a 25-mm Nuclepore filter with 5.0-,um pore size, and the filter, which retained the cells,was washed twice with 20 ml of ice-cold buffer. The filter was then air-dried, solubilized with Soluene-100(Packard), and counted by scintillation spectrophotometry in toluene-based scintillation cocktail. Specific activityof viruspreparations was determined byprecipitation of a sample of virus in
10% trichloroacetic acid on a Whatman GFA glass filter,digestedwithSoluene, and counted by scintil-lation. When radiolabeled virus particles were di-rectlyfiltered, less than 0.1% of the input radioac-tivity was retainedby the filter. The DNase lability oftrichloroaceticacid-precipitable counts in the fro-zer. viruspreparations was negligible.
Selection of resistant cells. A-9 cells resistantto
MVMinfection wereselected by allowingsurviving cells from an infectiontogrowoutandcloningcells from the resultant cell population. A-9 cells were
grown inmonolayer in F-11(minimalessential
me-dium, GIBCO) supplemented with 5% heat-inacti-vated fetal calf serum and 100 U ofpenicillinand 100 ,g of streptomycin per ml. The monolayers were infected as described for RT-7 cell infection (18). Afterlysis of most cells due to infection, a few survi-vors were noted and allowed to repopulate the origi-nal culture vessel over a period of 3 to 4 weeks. After regrowth of the monolayer, the culture now en-riched forcells resistant to MVMinfection was tryp-sinized with 0.25% trypsin in PBS (GIBCO) and diluted to 1 cell per 10 ul and seeded into microwell cloning plates at 10
sgl/well.
During outgrowth of the clones, each culture was monitored for the produc-tion ofviral proteins by hemagglutination assay, and only those cultures free of viral protein were subsequently tested for susceptibility to infection using a 50% tissue culture infective dose assay. Clones were also screened for virus binding using the assay systems described in Results.Electron microscopy. Normal A-9 cell monolay-ers in 60-mmculture dishes were rinsed in PBS at 4°C, and 106 110S MVM particles per cell were added in a total volume of 1 ml of PBS and allowed to adsorb at 4°C for 2 h. The monolayers were then washed in ice-cold PBS several times, fixed in 3% glutaraldehyde in phosphate buffer for 1 h at 4°C, and postfixed in 1% osmic acid. The monolayers wereembedded in Luft Epon mixture and sectioned with aDuPont diamond knife perpendicular to the plane of growth, as described by Anderson et al. (1). Electron micrographs were taken on a JEOL JEM 100B at 60-kV acceleration voltage.
RESULTS
Several conditions of thevirus-binding
reac-tion were examinedto optimizethe conditions
ofthe assay. Itwas
important
tominimizeup-take of virus into cells. Asuptake ofparticles by cells canbereducedby workingat low tem-peratures, the effectoftemperature onthe vi-rus-binding interactionswasexamined.InFig. 1, 105 virus
particles
percellwereadded to 2.2X 105A-9 cells in 1 ml of PBS at either4or21°C,
and the mixture was allowed to incubate for30
s or1, 3, 5, 15, 30, 60,or 120min. At the endof theincubationtime,thesamplewas filtered as
described
in Materials and Methods andwashed with ice-cold PBS, and the cell-bound counts per minute were measured. At both 4
and 21°C the reaction appears to consist of a
rapid
component within the first 30min and aslowcomponent that continuesfor at least 2h.
Adsorption times of upto 4 hhavebeen
exam-ined at 4°C, and the slow reaction appears to
continue
indefinitely
(notshown).
As tempera-ture appearstohaveonlyaslighteffect ontheJ. VIROL.
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BINDING SITES FOR MVM 213 201
9)
0
E9 E a.
9.
0
50
10
[image:3.504.272.417.260.443.2]30 60 90 120
Minutes
FIG. 1. Effect oftemperature on the binding of
MVMtoA-9 cells in suspension. A total of2 x105
cells in PBS, pH 7.2, were reacted with 104 [3H]thymidine-labeled 11OS MVMpercell for30sto
2hateither 21 or40C. The reactionwasstopped by filtration of the cell suspension through 25-mm Nu-clepore filters witha
5.0-Ium
pore size. Cell-associ-ated tritiumcountsperminuteareplottedversusthe time. Temperatureaffects therateof thereactionand the shape of the curve only slightly. Symbols: (0) 4CC; (0)21 C.extentof virus binding, subsequent binding
ex-perimentswereperformedat 40C for 2 hasthe
standard conditions.
Little ifanyof the virus boundtocellsat40C
appears tobe takenupduringthe 2-h
incuba-tion period,as78% of the cell-associatedcounts
can be removed by a brief wash (5
min)
inCa2+,Mg2+-free PBS with 0.001 M EDTA(Table 1). Treatment of this sort has no observable
effects on plating efficiency of the cells. Thus,
the bindingassay appearsto measure
primar-ily surface attachment and isnotsignificantly complicated by uptake into cells. This
conclu-sion is supported by the fact that virus previ-ously boundtothe cellsurfacecan be competi-tively displaced by subsequently added unla-beled virus (Fig. 2). In this experiment a sub-saturating amount of labeled virus (104
parti-clespercell) wasallowedtobindtocellsat 40C
for2 h. Increasing concentrations of unlabeled
virus were then added to the cell suspension.
After1h, theamountof residual label boundto
the cellswasmeasured. ItcanbeseenfromFig.
2, curve
B,
that theamountof cell-bound label remains constant until the input multiplicity exceeds 5 x 105 particles per cell. However, once this saturation level has been exceeded,the additional unlabeled viruseffectively
com-petes with thelabeled virus for attachment to
the cell surface. These results indicate that binding of virustothe cellsurface ismostlikely
areversible reaction and thatamajor portionof
the virus isprobably bound atthe cell surface under theconditions of the bindingassay.
Acritical question in measuringanybinding
TABLE 1. Effect of wash treatment on amount of virus bound tocellsa
cpmbound
Treatment Avg % oftotal
Sample 1 Sample 2
Control 1,248 1,186 1,217 100
EDTA wash 219 334 276 22
a Four samples each containing 2 x 105 cells
sus-pended in1ml ofPBS at40Cwerereacted with 105
[3H]thymidine-labeled MVMparticles per cell for 2 h. At the end of the incubation time the samples were filtered and washed as described in the text. Two samples were then washed for 5 minutes (slow filtration) inCa2OMg2+-freePBS with 1 mM EDTA. A total of 78% of the cell-associated counts was washed off by this treatment.
15
0
N 10 \
0
0~~~~~\
tA
101lo10"
lo12
Io13
(unlabeled]
FIG. 2. Curve of competition between binding la-beled and unlala-beledMVM;A-9 cells suspended in PBS at 40C were reacted with a
fixed
quantity of[3H]thymidine-labeled MVM (2.4 x109particles).In the first case (curve A, 0) the labeled virus was mixed with increasing amounts of unlabeled virus prior toadsorption to cells (2 x 105). Inthe second instance(curve B, 0)thelabeled virus was allowed to adsorb tothecells(1.7 x 105)for2h, and increasing amounts ofunlabeled virus were subsequently al-lowed to incubate with the cells for 1 h prior to
filtration.The arrow indicates the point at which the input multiplicity reached 5 x105 virusparticles per cell.
to cellsurfaces is the extent to which the reac-tion is specific. Generally, specific attachment is limited and therefore saturates as the amount of input
material
increases. Inaddi-tion, attachment of labeled particles to specific sites on the cell surface can be competed by unlabeled particles, and this is shown for MVM
binding
inFig. 2, curve A. In this case a fixedamountoflabeled virus was added to the cells
along
with increasing amounts of unlabeledVOL. 24, 1977
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particles. It can be seen that the amount of
bound radioactivity beginstodecline when the total input virus approaches5 x 105input
parti-cles percell. Dueto the very high amountsof
unlabeled virus required, we are unable to
demonstrate complete competition. However, the results indicate thatgreaterthan 75% of the viral binding can be competed by unlabeled
virusand is specific by this criteria.
It has been previously noted that parvovirus binding to erythrocytes (hemagglutination) and cellular debris could be minimizedathigh pH (9).Consequently, the dependence uponpH
of MVMbindingto A-9 cellswasexamined by
usingaspectrumof organic bufferstocoverthe
pH range from 6.5 to 9.0 (Table 2). Figure 3 shows that the binding reaction has an opti-mum pH occurring near neutrality. These
ex-periments were repeated using inorganic
buffers(datanotshown), and the pH optimum
wasbetween 7.0 and 7.2 for the binding
reac-tion.
A second important indication of specific binding of virustocellularreceptors is the
cor-relation of infectivity with virus binding. To further demonstrate thatwe are measuring a
specific binding ofvirus to cells, we isolated a
series of A-9 cell clones that wereresistant to
virusinfection and tested them for the loss of virusbinding.
Resistance A-9 cells.Resistanceof A-9clones
to MVM infection was measured by using a
modified 50% tissue culture infective doseassay
for the production of viral hemagglutinin. Ta-ble 3 shows the data forcomparisonof infection
of control A-9 cells and the clonal derivative
designated 8-E. The cellswere infectedby ad-TABLE 2. Buffer solutions usedtogenerateFig.3a
Concn
Buffer" pK. used pHused
(mM)
BIS Tris 6.46 20 6.50
PIPES 6.80 10 6.75;7.00
BES 7.15 20 7.25
TES 7.50 20 7.50
HEPES 7.55 20 7.75
HEPPS (EPPS) 8.00 20 8.00
Tricine 8.15 20 8.25
Bicine 8.35 20 8.50
Tris 8.30 20 8.75; 9.00
aEach buffer solution also contained0.9%NaCl,
7 x 10-4M CaCl2,and 5 x 10-4MMgC12.
bBIS, N,N-methylenebisacrylamide; PIPES,
pi-perazine-N,N'-bis(2-ethanesulfonic acid); TES, N-tris(hydroxymethyl)methyl-2-aminomethane-
sul-fonicacid; HEPES, N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid; BES, N,N-bis(2-hydroxy-ethyl)-2-aminoethane sulfonic acid; HEPPS, N-2-hydroxyethylpiperazinepropanesulfonic acid.
'5
-0
E 10
-5
-6.5 7.0 7.5 8.0 8.5 9.0 pH
FIG. 3. pH optimum for MVM binding reaction; 2 x105 A-9 cells suspended in various buffer combina-tions (listed in Table 2) were reacted with 105 [3H]thymidine-labeled viral particles per cell at40C for2h. The suspensions werefilteredasdescribed in the text, and the cell-associated radioactivity was determined. There is pH optimum at pH 7.0. Each point is the average ofthree determinationsplusor
minus therange.
sorption of dilutions of 110S
MVMstock in PBSat
370C
for
2h. The control A-9 cellsproduce
theexpected dilution-dependent
curve in terms ofhemagglutination activity (Fig. 4). By compari-son, it appears
that the
8-Ecells require
a5-log-unit-higher
concentration of input virus thando the control
A-9cells
toproduce the
sameamount
of
hemagglutinin. The
50% tissue cul-ture infectivedose
curvefor
thenormal
cellspeaks
in themiddle, demonstrating
the cellgrowth
dependence
ofviral
production.
At thehighest multiplicity of infection
alarger
per-centage
of
cells
isinitially infected and
conse-quently inhibited from further cell division.
Cells infected
at alower
multiplicity of
infec-tion
can gothrough several cell
growth cycles
prior
tospread of the infection
toall of
the cells.
The infection
wasallowed
toproceed for
4days
to
confluency, explaining the lower
titersof
viral
protein at thelowest multiplicities of
in-fection.
In
Fig. 5 virus-binding saturation curvesgenerated
inparallel
onnormal A-9andresist-ant 8-E cells are shown. The A-9 curve is bi-phasic, consisting of a specific,
saturable
com-ponent saturating atapproximately
5 x 105 virus particles bound per celland an apparently nonspecific insaturable component. Virus bind-ing tothe 8-E cells is monophasic andinsatura-ble under this
condition, mimicking
thenon-specific
portion of the A-9virus-binding
curve.The 8-E cells appear to have lost the specific virusreceptor present in 5 x 105copies per A-9 cell.
Figure 6 is a saturation curve comparing the
permissive
A-9 cell line and thenonpermissive
L1210 line. In the case of the permissive A-9
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[image:4.504.294.457.61.212.2] [image:4.504.68.259.482.597.2]BINDING SITES FOR MVM 215 TABLE 3. Hemagglutination activityproduced in a tissue culture infectivity assay run on A-9 and 8-E cells in
parallels
Dilution of infectious Hemagglutinin production
stock A-9 Mean 8-E Mean
10-2 64; 64; 128 85 2;4;4 3.3
10-3 128; 128; 256 170 0; 0; 0 0
10-4 128; 512; 512 384 0; 0; 0 0
10-5 64; 64; 64 64 0; 0; 0 0
10-6 32; 8; 8 16 0; 0; 0 0
10-7 2;2;2 2 0; 0;0 0
aA total of 105 cells were seeded into 35-mm culture dishes. Dilutions of stock MVM were adsorbed in 0.5
ml of PBS at
370C
for 2 h. The cultureswere subsequently fed with growth media (see text) and refed daily. Four days after infection, mock-infected control cultures of both cell types had reached confluence. The lowest three dilution samples exhibited gross cytopathology in the A-9 cultures, whereas no growth inhibition or cytopathic effect was evident in any 8-E cultures. At this time cells wereharvested into 0.01 M Tris-0.005 M EDTA (pH 9.0) buffer andlysed by sonic treatment. Viral protein was assayed by thestandard hemagglutination assay.5
cam 4
x 3
2 .
in
I0
x
[image:5.504.50.448.97.189.2].i
dilution
FIG. 4. Tissue culture infectivityassayperformed oncontrol A-9 cells andaresistant cloneofA-9 cells
designated 8-E: 105 A-9 cells or8-E cells in
mono-layerwere infected with the dilutions ofstock virus
shownby adsorptioninPBSat370Cfor2 h.After4
days in growth media all ofthe 8-E cultures and
severalofthe A-9 cultures had reachedconfluency.
Atthistime, each samplewasharvestedinto 0.01 M
Tris-O.005 M EDTA (pH 9.0) buffer, sonically treated,andassayed forviralprotein bythe hemag-glutinationassay.The resultsareshown inTable3,
andthemeanof the triplicatemeasurementisplotted
inFig.5.8-Ecells produced measurable quantities of hemagglutinin only atthe highest concentration of
virus used, whereas A-9 cellsproduced
hemaggluti-nin atallconcentrations of input virus used.
Sym-bols: A-9 (a);8-E (0).
cells, again there is abreak in the saturation curve at between 5 x 105 and 7 x 105 viral
particles boundper cell, reflecting the number
of specific binding sites. The nonpermissive
L1210 line shows little appreciable binding in
the concentration range used in this
experi-ment. Much lower levels of "specific" binding
mayexistfor theL1210cell line, but it is inno
waycomparable tothe A-9 system. The L1210 saturationdata whenplotted on alower scale
1 2 3 4 5 6
I.M.x 10-6
FIG. 5. ComparisonofMVMbinding to-control
A-9cells andtoresistant cloned derivativesofA-9cells
designated
8-E;
2 x104A-9or8-E cellssuspendedin PBS at40C
werereacted with the indicatedmultiplic-ityof[3H]thymidine-labeled MVMparticlespercell (inputmultiplicity [IM.])for2h.Sampleswerethen filtered, and the cell-associated radioactivity was measured and converted to bound multiplicity
(B.M.)asdescribed in thetext.Atall concentrations
ofinput virus,filtration of cell-free controlsamples
resulted in retentionofless than0.1% ofthe input
radioactivity, whereassampleswith cellsretained up
to75%.Thecontrol A-9 cells bind virusinabiphasic
manner, with saturation ofthefirstcomponent oc-curringatabout5 x105MVMparticlespercelland the second componentnotsaturable under these con-ditions. Symbols:A-9 (0); 8-E (a).
(one log unit) generate a monophasic curve similar to the 8-E curve (not shown). This indi-cates
that
the L1210 cells also bind virus with nonsaturating kinetics. The reaction is, how-ever, of even lower affinity than observed for virus binding by 8-E cells.In view
of the
reportsthatspecific
tissuesaresusceptible
toparvovirus
infectionduring
the courseof
development (3, 12),
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[image:5.504.73.219.266.400.2] [image:5.504.252.443.266.409.2]in 0
2
1 2 3 4 5 6
I.M. x 106
FIG. 6. Comparison ofMVM binding to
permis-sive A-9 cells andtononpermissiveL-1210 cells: 2 x
105 cells (A-9 orL-1210) suspended inPBSat40C
were reacted with the indicated number of [3H]thymidine-labeledMVMparticlespercell(input multiplicity [IM.]) for2 h.Samples were then fil-tered,and thecell-associatedradioactivity was
mea-sured and convertedtoboundmultiplicity (B.M.)as
described in thetext.Symbols:A-9 (0);L-1210 (0).
Each point is the average ofthree determinations
plusorminus therange.
to
determine
ifthe number of viralattachment
sites
changed
in cellsundergoing
"differentia-tion." Friend-745erythroleukemia
cells canbeinduced to differentiate and produce hemoglo-bin in culture with Me2SO
(6).
These cellsarepermissive for MVM
growth
inuninduced cul-tures (16a). Friend cells appear to be a stemcell in the erythropoietic series, which
ul-timately leads
in vivo to formation ofmouseerythrocytes.
Theseerythrocytes bind and
areagglutinated by
the virus. In viewof these
facts,
wewished
to determine if thenum-ber
of virus-binding
sites per cell changesduring
the induceddifferentiation of these cellsin
culture.
Aculture of uninduced Friend cells
was
split
into twoequal volumes
offresh
me-dia,
oneofwhich received 1.8%Me2SO.Induc-tionwasmeasuredby counting the percentage
ofbenzidine
(hemoglobin)-positive
cells in each culture.Initially
both cultures wereless than1%
benzidine
positive, and the uninducedcul-ture
remained
assuch
throughout successivedays.
The induced culturesteadily
increased inthe
proportion
of benzidine-positive
cells,
reaching
apeak of
50% on day 4. Cells wereharvested and
suspended
incold
PBS,and the
virus-binding assay as described for A-9 cells
above (see also Materials and
Methods)
wasused to titrate the number of specific binding sitespercell inthe
parallel
cultures.Figure
7shows that both induced and
uninduced
cul-tures possess
about
1.5 x 105saturable binding
sites per cell,
and no difference between the twois
detectable with our methods.
Electron
microscopy. In an attempt to
visu-alize the virus-binding sites on the surface of
the cell, A-9 cells in monolayer
wereexposed
to106 virus
particles per cell
inPBS at
4VC for 2 h.
The monolayers were rinsed and prepared for
electron microscopy as described in Materials
and Methods. Due to virus adsorption to the
substrate
(unpublished observations) as well as
equilibrium considerations (see
Fig. 5 and 6),fewer than
5 x105
particles are bound per cell
under these conditions. MVM can be seen to
bind to at
least three morphologically distinct
regions of the surface (Fig. 8 and 9). First of
all, clusters of virus,
aswell
assingle particles,
can
be
seenon
the surfaces of numerous
filipo-dia. Small
patches of particles, as well as single
particles,
canalso be
seenscattered
overthe
apparently unspecialized regions of the cell
sur-face. In
addition,
virus
particles can
be
seenlocalized
inspecialized clefts
inthe cell
mem-brane. These clefts are characterized by a
prom-inent submembranous
thickening
and outersurface glycocalyx and
appear to be
endocy-totic regions
of the cell surface (1, 7).
5-4
-0
I 2 3 4 5 6
I.MX 10 6
FIG. 7. Comparison of saturation binding
of
MVMtoinduced anduninduced Friend-745 erythro-leukemiccells;2 x 105inducedoruninducedFriend-745 cells (Fig. 8)
suspended
inPBS at40C were reacted with the indicated numberoftHithymidine-labeledparticles (inputmultiplicity [I.M.])
for
2 h.Sampleswerethenfiltered,and thebound
multiplic-ity (B.M.) was measured as described in the text. Both induced and uninduced cultures bind virus withabiphasic curve, which goesthrough
atransi-tion at approximately1.5 x 105saturable
binding
sitesper cell. Symbols: uninduced Friend-745 cells
(0);induced Friend-745 cells (-).Each
point
is the average ofthree determinations plus or minus the range.on November 10, 2019 by guest
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[image:6.504.68.258.59.240.2] [image:6.504.275.465.362.526.2]VOL.24,1977~~~~~BINDINGSITES FOR MVM
217
Is,~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~sy
8
a~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~a
-A>~~~~~~~~A
.4.
wt~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~'
4~~~~~~~
46%~~~~~~~
e~~~~~~
b
~~~~~~~~~~~A*~~~~~~~~0
.-Jt
C
., Q 6
4.0-Z... '. I-A
)e
454'
*'~~~~~~~~~~~~~~407`
nA ~ ~ ~ ~
'~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4
FIG. 8. Electron micrographs ofthe surface ofA-9 cells fixed-after a2-h exposure to 106JiOS MVM
particlespercell at4'C. (a) Low magnification ofA-9 cellshowingseveral scatteredpatches ofadsorbed MVM. Thearrows indicateafewsuchpatches. The crossedarrowspointoutclustersofvirionsadheringto
filipodia. x 18,000. (b) High-magnification micrograph showingalarge patch ofMVMboundto the cell
surface. The sectionwasapparentlycutperpendiculartotheplane ofthe cell membraneattheregionofthe viruspatch.Thisareaofthe membraneappears toberelatively unspecializedmorphologically.Anadditional
patch ofviruscanbeseentocontinuefromtheflatcellsurfaceupthe stalkofafilipodium(arrow) x60,000.
(c) Electronmicrograph showingvirusboundtofilipodia(arrow)as wellasatangentialsection througha
viru8 patchillustratingtheparacrystalinnatureofsuchpatchesatthe cellsurface(crossedarrow). x45,000.
rt.N
V. I'
4".
VOL.24,
1977on November 10, 2019 by guest
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[image:7.504.46.447.81.595.2]218
LINSER, BRUNING, AND ARMENTROUT9a
41
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c;
,
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.N
V r f
4;
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^
.- 4
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;IV t
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tv'Nsm*Air~~~I
*r, fA s>rz",T ~ .
i
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;NR i i .~ S X
--SpsF;
FiG. 9. Electronmicrographs of the surface ofA-9cellspreparedasinFig. 8. (a) Micrographshowing
virusboundto unspecializedregions of surface membraneas wellas toa morphologicallydistinguishable
region ofmembrane. Thisspecializedregion ischaracterized byaprominentthickeningonthe inside(arrow
heads) and a less clearly visible glycocalyx on the outside surface of the plasmalemma. x60,000. (b)
Micrograph again showing virus particles adsorbed to a specific membrane region characterized by a
prominent submenbranous thickening(arrowheads) and lightly visibleouterglycocalyx (arrow). x65,000.
(c)MVMparticlesadsorbedtounspecialized regions of cell surfaceingroups(arrow)andassingleparticles
(arrow heads). x60,OOO.
,sk
.
rf,
J. VIROL.
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[image:8.504.68.466.75.588.2]BINDING SITES FOR MVM 219
These
results demonstrate
that
when the
viral
receptor
sites are in excess,
virus is
ob-served
on
several regions of the cell surface.
Further
studies are in progress to delineate the
processes by which the bound virus enters the
cell.
DISCUSSION
Utilizing
aradiolabeled virus
probe,
wehave
characterized and quantified the number of
"specific"
MVM
binding sites
onthe surface
of
several murine cell lines. The A-9 derivative of
mouse
L-cells
(13)
binds
virus
with
abiphasic
saturation
curveand
apH optimum of 7.2
at4VC.
The
biphasic
saturation
curve canbe
bro-ken into a
rapidly saturable and
an
apparently
insaturable reaction.
There
are
approximately
5 x 10, saturable binding sites on A-9 cells.
Friend-745
erythroleukemic cells, another
mu-rine
cell
line
permissive for
MVM
growth
(16a),
also bind virus in a biphasic reaction
with
approximately
1.5 x 105saturable
bind-ing sites per cell. The mouse
lymphoid cell line
L1210, however, is not
susceptible to MVM
infection and binds little
virus. A cloned
variant of the A-9 cell line
selected for
re-sistance to infection
with MVMwas also
de-veloped and shown to
apparently lack the
saturable component of the biphasic binding
curve.
These results indicate that the
satur-able
and,
consequently, specific binding
sites
on
A-9
cells
aredirectly
involved in the
infec-tious process.
Most
of the cell-associated
counts
following
abinding
reaction
at4VC
are atthe surface
of the
cell and
notinternalized.
About
80%of the
cell-associated
counts canbe washed off the cell
with
abrief exposure
toCa2+,Mg2+-free
PBS
containing
0.001 MEDTA.
After
a2-h
incuba-tion
with
subsaturating
quantities of labeled
virus, at least
45%of the
counts
can
be
removed
from
the cell
in a
1-h
chase
with excess
cold
virus.
This last observation suggests that the
binding
of MVM
at4VC is
reversible
and will
consequently approach equilibrium.
Rough
estimates
of the initial
rateof
attach-ment
werecalculated from the
slope
of
the
binding
curves(K)
at 30s,
asdescribed
by
Lonberg-Holm
and
Whiteley
(16). At
4VC
the
value equals 2.8
x 10-7cmVmin,
and at
210
C Kequals
4.4 x 10-cm:/min. This
represents
anincrease
in Kby
afactor of
1.33/100C.
This
isvery close
tothe
predicted effects of
tempera-ture on the diffusion coefficient for
particles
this size
(i.e.,
1.30/100C)
(16, 22).
Thus, the
difference seen between reactions
occurring at
4and
21'C seem tobe due
primarily
tothe
change
inparticle diffusion
rates.Also,
sincethe
phase
transitionfor
membrane
lipids occurs
at
18'C (K.
Lonberg-Holm
and L.
Phillipson,
in
Tiffany and Blough, ed., Cell Membranes and
Viral Envelopes, in press), it does not
appear
that
the binding of MVM as measured here
re-quires free
lateral movement of receptors,
contrary to what has been reported for
adeno-virus
(17).
The
binding of
several different
picornavi-ruses, as
well as adenovirus, is affected
some-what more
by temperature than MVM
binding
(16),
although there
is
considerable
variability
(2, 11). The
initial
rates
of
attachment
for MVM
calculated
at
4and
21°C
areconsiderably
morerapid than for
picornaviruses
in
general (i.e.,
10-8 to 10-9
cm:'/min
at 30 to37°C)
(16).
The
theoretical
maximum
rateof
1.7 x 10-7 cm3/
min for
picornaviruses (16) is
also
somewhat
slower than
observed for
MVM.
This may
re-flect the
crudeness of
our rate measurements.On the
other
hand,
this may
be
due
atleast in
part to
the
considerable
differences
that
exist
between the
picornavirus-receptor
relationship
and
MVM-receptor interactions.
Picornavi-ruses
bind to
104receptors
per
cell,
whereas
MVM
receptors appear to be
about
50times
asnumerous,
which
would
increase
the
rateof
parvovirus
binding by
increasing
the number of
effective cell-virus collisions. The picornavirus
receptor reaction has been generally reported
as
being largely
irreversible.
MVMbinding
measured at 4°C is, however, readily reversible
and
apparently defined by second-order
equilib-rium
kinetics.
The
virus
preparations used to measure the
above
findings
were
well suited for these
pur-poses.
The
virus
was
radiolabeled
with
PH
thymidine
toavoid such
changes
in
the
surface properties of the virus as might be
pro-duced by techniques such as radiolabeling with
iodine. The virus is isolated in velocity
gra-dients
to assure
that the particles are
monodis-perse
when
introduced into a reaction, an
im-portant consideration in view of the tendency
for
MVMto
aggregate
when
placed into a
high-salt environment such as CsCl equilibrium
density
gradients. Once the virus has been
placed
into
physiological salt concentrations as
in
the binding reaction mixtures, it is
impossi-ble
tocontrol aggregation, and this may
con-tribute
to the
nonspecific
component of the
binding
reaction.The
purity of the virus
probe was confirmed
by sodium dodecyl
sulfate-discontinuous gel
electrophoresis (18). Only viral proteins are
ob-served
inthe 110S material from the
sucrose
velocity gradients. All of the
radioactivity is
acid
precipitable and resistant to DNase. These
observations
indicate that
all of the the
cell-associated
radioactivity after a binding
reac-VOL. 24, 1977
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220
tion
indeed represents
adsorbed
or
internalized
virus.
The results
of the binding
of virus to
induced
and uninduced Friend-745 cells indicate that
the
virus-binding
sites do not
change
on
the
surface, although during
Me2SO-induced
differ-entiation the cell surface
undergoes several
al-terations
with the appearance
of
newantigens
(8).
Whereas the viral attachment sites
arepresent in
the extreme
form
of the
differen-tiated cell in vivo,
the
mouse
erythrocyte,
our
results indicate
that
virus-binding
sites are
probably
retained throughout the
differentia-tion process in
about the
samenumbers
per
cell
as present on
the
precursor
cells.
Furthermore,
in
light
of these
results
it
is
unlikely that the
uninduced cultures
of Friend-745 cells support
growth of
MVM
due
tothe presence of
afew
spontaneously induced
cells that
have
viral
re-ceptors among a
large
population
of
resistant
cells lacking receptors.
It is worth noting
that
an
erythroid
tumor
line possesses virus-binding sites whereas the
lymphoid
tumor
line
(L1210)
appears
tolack
receptors.
The difference between
lymphoid
and
erythroid
cells in
termsof MVM infection
susceptibility has
been
previously
noted
by
Miller et al. (16a).
Preliminary
electron
microscopic
examina-tion
of
virus
bound
to
A-9
cells in
subsaturating
quantities
reveals
binding
toseveral
morpho-logically distinct regions
of the cell surface.
A
comprehensive study
utilizing
specific
virus
la-beling
is
under
way
toshed
further
light
onthe
nature
of the
specific, infection-related
binding
sites.
The
data we have presented on the binding of
MVM to
cells
canbe
explained by
asimple
model: cells sensitive
toviral infection bind
virus to
specific
sites
onthe
surfaces;
somevirus is
also
bound
nonspecifically,
but
such
binding
is very
inefficient
in
causing infection
of the cell.
However,
it
should be noted
that
ourviral
probe
consists
of the
full virus
class
of
particles, which
is
nothomogeneous.
The
110S
virus consists of at least two classes of
particles:
a
low but variable
percentage (5
to25%) of
adense
(1.46
g/cm3
inCsCl) precursor particle
and
ahigh
percentage of
alighter
(1.42
g/cm3
inCsCl) product particle
(4, 18). Some evidence
has been presented which might indicate that
the
minor, dense
species has
apoor
affinity
for
cells
compared
with the
binding
of the
major
species (4).
In ourexperiments,
wehave
ob-served
up
to 60 to 65% ofthe
input
particles
bound
tocells
when sites are in excess.These
results would
support
theidea
that ourbinding
assay
measures thebinding
of thepredominant
1.42-g/cm3 density class particles. However,
re-cent results indicate that both classes of viral
particles are equally rapidly bound to cells
un-der our assay conditions (P. Linser and R. W.
Armentrout, in D. C. Ward and P.
Tattersall,
ed., Parvoviruses, in press). The fact
that the
viral preparations used in the binding assay
are not
homogeneous limits the
conclusions
that can be derived from the kinetic data.
Nevertheless, the heterogeneity of the viral
particle preparations does not affect our
conclu-sion
that specific cell surface receptors are
re-quired for efficient infection of cells.
ACKNOWLEDGMENTS
Wegratefullyacknowledge helpfuldiscussions with Pe-terTattersall and David Ward duringthe courseof this work. R. Morrisprovidedexperttechnical assistance.
This investigation wassupported byPublic Health Serv-ice grants 1 K04CA 00134and5R01 CA-16517awardedby the National CancerInstitute, grant 1-396from the Na-tional Science Foundation-March ofDimes, and a grant from theUnitedFundHealth FoundationofCanton,Ohio.
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